Pneumolysin mutants and pneumococcal vaccines made therefrom

ABSTRACT

Mutants of pneumolysin that are non-toxic by reason of amino acid substitutions have been constructed. These mutants elicit an immune response in animals that is reactive to wild-type pneumolysin. The invention also encompasses vaccines for humans based on these mutants, including vaccines comprising conjugates with pneumococcal capsular polysaccharides.

This application is a continuation-in-part of application Ser. No.08/290,501, filed Aug. 15, 1994, now abandoned, which acontinuation-in-part of Ser. No. 07/721,444, filed Jul. 30, 1991, nowabandoned, which was the 35 USC §371 national phase of Internationalapplication PCT/AU89/00539, filed on Dec. 15, 1989, which designated theUnited States of America.

This invention relates to mutants of the toxin pneumolysin andpneumococcal vaccines based on these mutants.

BACKGROUND OF THE INVENTION

Streptococcus pneumoniae (pneumococcus) is an important pathogen,causing invasive diseases such as pneumonia, meningitis and bacteremia.Even in regions where effective antibiotic therapy is freely available,the mortality rate from pneumococcal pneumonia can be as high as 19% inhospitalized patients and this increases to 30-40% in patients withbacteremia. These high mortality rates have been reported in the U.S.A.where pneumonia, of which S. pneumoniae is the commonest cause, is thefifth ranking cause of death. Indeed, pneumonia is the only infectiousdisease among the top ten causes of death in that country. In the UnitedStates mortality rates for pneumococcal meningitis range from 13-45%. Indeveloping countries, in excess of 3 million children under the age of 5years die each year from pneumonia, and again S. pneumoniae is thecommonest causative agent. S. pneumoniae also causes less serious, buthighly prevalent infections such as otitis media and sinusitis, whichhave a significant impact on health-care costs in developed countries.Otitis media is especially important in young children; sinusitisaffects both children and adults.

In the late 1970's, a vaccine was licensed for the purpose of preventingserious infections, especially bacterial pneumonia and for protectingcertain groups, such as splenectomized individuals and young children,who are particularly susceptible to fulminating pneumococcal disease.The vaccine is composed of purified capsular polysaccharides, which arethe predominant pneumococcal surface antigens. However, each serotype ofS. pneumoniae (of which there are 83) has a structurally distinctcapsular polysaccharides, and immunization with one serotype confers noprotection whatsoever against the vast majority of the others. Thevaccine currently licensed in Australia contains polysaccharidespurified from the 23 most common serotypes, which account forapproximately 90% of pneumococcal infections in this country.

Protection even against those serotypes contained in the vaccine is byno means complete, and there have been several reports of serious, evenfatal infections occurring in vaccinated high-risk individuals. Theefficacy of the vaccine is poorest in young children, and severalstudies, including one conducted in Adelaide, have shown that theexisting formulation has little or no demonstrable clinical benefit inthis group. This apparent failure cm the vaccine appears to be relatedto the poor immunogenicity of certain pneumococcal polysaccharides inchildren under 5 years of age. We have shown that the antibody responseis particularly poor to the five serotypes which most commonly causedisease in children (types 6, 14, 18, 19 and 23). Indeed, the antibodyresponse to these pneumococcal polysaccharides only approaches adultlevels in children over 8 years of age at the time of vaccination.

In view of this, a vaccine, including antigens other than the capsularpolysaccharides seems to be required to protect young children frompneumococcal infection. One such antigen could be pneumolysin, a proteintoxin produced by all virulent S. pneumoniae isolates.

Immunization of mice with this protein has been found to confer a degreeof protection from pneumococcal infection. However there is a difficultyin that pneumolysin is toxic to humans. Thus pneumolysin included in avaccine must therefore be substantially non-toxic. However, therendering of a pneumolysin non-toxic by most currently employed methodswould be likely to alter the basic configuration of the protein so as tobe immunogenically distinct from the native or wild-type pneumolysin. Animmune response elicited by an altered protein that is immunogenicallydistinct from the native pneumolysin will have a decreased protectivecapacity or no protective capacity. Thus the difficulty is to produce analtered pneumolysin that is non-toxic and at the same time sufficientlyimmunogenically similar to the toxic form to elicit a protective immuneresponse.

An altered pneumolysin with the above characteristics can then be usedin a number of ways in a vaccine. Thus the altered pneumolysin may beused by itself to immunize, or alternatively the altered pneumolysin maybe conjugated to pneumococcal polysaccharide, or alternatively may beincluded in a vaccine wherein pneumococcal polysaccharides may beconjugated to another protein and the altered pneumolysin is present ina non-conjugated form only. Alternatively, pneumococcal polysaccharideand pneumolysin may both be used in an unconjugated form.

DESCRIPTION OF INVENTION

In a broad form therefore the invention may be said to reside in analtered pneumolysin being substantially non-toxic and being capable ofeliciting an immune response in an animal susceptible to wild-typepneumolysin.

Preferably the altered pneumolysin has reduced complement bindingactivity as compared to wild-type pneumolysin. Reduction in thecomplement binding activity results in less inflammation at the site ofadministering the vaccine.

Preferably the altered pneumolysin has reduced Fc binding activity ascompared to wild-type pneumolysin. Reduction in the Fc binding activityresults in less inflammation at the site of administering the vaccine.

Preferably the altered pneumolysin is altered by reason of one or moreamino acid substitutions relative to wild-type pneumolysin.

The pneumolysin may be altered in that the amino acid present at any oneor more than one of residue sites 367, 384, 385, 428, 433 or 435 ofwild-type pneumolysin are replaced, removed or blocked.

In a further form the invention could be said to reside in a vaccineincluding an altered pneumolysin, said altered pneumolysin beingnontoxic and being capable of eliciting an immune response in an animalbeing reactive to wild-type pneumolysin.

Preferably the vaccine comprises capsular polysaccharide materialconjugated with the altered pneumolysin.

The capsular material may be derived from any one or more of theStreptococcus pneumoniae serotypes 6A, 6B, 14, 18C, 19A, 19F, 23F, 1, 2,3, 4, 5, 7F, 8, 9N, 9V, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F.

In this embodiment, serotypes which are commonly associated with diseasein children, and to which children generally have a poor immuneresponse, may be specifically targeted (i.e. Danish serotypes 6A, 6B,14, 18C, 19A, 19F and 23F). Other common serotypes contained in thepresent 23-valent Merck Sharp and Dohme vaccine (Pneumovax 23) however,could also be used to synthesize conjugates (i.e. types 1, 2, 3, 4, 5,7F, 8, 9N, 9V, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F) or indeed anyother serotype. Conjugation of any pneumococcal polysaccharides to theprotein carrier ensures good T-cell dependent immunogenicity inchildren, such that protective levels of anti-polysaccharide antibodyare produced.

The combination of the altered pneumolysin together with the capsularmaterial will ensure an extra degree of protection, particularly againstserotypes of S. pneumoniae whose polysaccharides are not incorporated inthe existing vaccine formulations.

The vaccine is preferably administered by sub-cutaneous injection, withor without an approved adjuvant, such as alumina gel.

In another form the invention could be said to reside in a recombinantclone including a replicon and a DNA sequence encoding an alteredpneumolysin, said altered pneumolysin being non-toxic and being capableof eliciting an immune response in an animal susceptible to wild-typepneumolysin.

In yet another form the invention could be said to reside in a method ofproducing an altered pneumolysin including the steps of purifying saidaltered pneumolysin from an expression system including a recombinantclone with DNA encoding an altered pneumolysin said pneumolysin beingsubstantially non-toxic and being capable of eliciting an immuneresponse in an animal susceptible to wild-type pneumolysin.

Preferably the expression system is a culture of a host cell including arecombinant clone with DNA encoding the altered pneumolysin.

In another form the invention could be said to reside in a method ofproducing a vaccine including the step of amplifying-a recombinant cloneencoding an altered pneumolysin, inducing transcription and translationof said cloned material, the purification of altered pneumolysin, andthe step of conjugating the altered pneumolysin with a capsularpolysaccharide, the altered pneumolysin having substantially reducedtoxic activity as compared with wild-type pneumolysin.

For a better understanding of the invention specific embodiments of theinvention will now be described with reference to diagrams wherein:

FIG. 1 is the DNA sequence of the gene encoding wild-type pneumolysin(SEQ ID NO:1);

FIG. 2 is the DNA sequence (SEQ ID NO:3) of an altered gene encodingwild type pneumolysin used for cloning the pneumolysin gene into anexpression vector,

FIGS. 3a and 3 b show the amino acid sequence (SEQ ID NO:2) of thewild-type pneumolysin as derived from the DNA sequence of the geneencoding the wild type pneumolysin,

FIGS. 4a and 4 b show the amino acid sequence (SEQ ID NO:2) ofpneumolysin showing amino acid substitutions introduced by site directedmutagenesis,

FIG. 5 is a physical map of the pGEMEX-1 vector encoding for mutantpneumolysin,

FIG. 6 shows the electrophoresis of the purification run forpneumolysoid,

FIG. 7 shows the levels of total immunoglobulin in serum of MF1 miceafter various vaccinations, and

FIG. 8 shows the levels of anti-PL immunoglobulins of different isotypesin serum of MF1 mice, after vaccination with a certain pneumolysoid.

Recombinant DNA techniques have been used to construct non-toxicpneumolysin derivatives suitable for administration to humans. Toachieve this, the S. pneumoniae gene encoding pneumolysin was clonedinto Escherichia coli and its complete DNA sequence determined. The DNAsequence is shown in FIG. 1 and the derived amino acid sequence is shownin FIGS. 3a and 3 b.

Three regions of the pneumolysin gene were subjected tooligonucleotide-directed mutagenesis. The first region encodes aminoacids 427-437 in the protein sequence, and is indicated by an underlinein FIG. 3b. This 11 amino acid sequence shows absolute homology withsimilar regions in other related thiol activated toxins thus is thoughtto be responsible for the hemolytic activity and hence toxic activity ofthe toxin. The other two regions encode amino acids 257-297 and aminoacids 368-397 and are also indicated by an underline in FIG. 3b. Thesetwo regions of the toxin have substantial amino acid sequence homologywith human C-reactive protein (CRP), and by inference therefore, arethought to be responsible for the ability of pneumolysin to bind the Fcregion of immunoglobulins and to activate complement. Fifteen separatemutations in the pneumolysin gene, resulting in single amino acidsubstitutions, were constructed, as shown in FIGS. 4a and 4 b. In aneffort to maintain the structure of the altered pneumolysin,conservative substitutions were made, so that amino acids aresubstituted with amino acids of a similar nature.

For the region invoked in hemolytic activity, Cys₄₂₈→Gly, Cys₄₂₈→Ser,Trp₄₃₃→Phe, Glu₄₃₄→Asp and Trp₄₃₅→Phe each reduced hemolytic activity by97%, 90%, 99%, 75% and 90% respectively. The other mutations in thatregion Cys₄₂₈→Ala, Glu₄₃₄→Gln and Trp₄₃₆→Phe did not affect hemolyticactivity. Mutating a separate region of the toxin thought to beresponsible for binding to target cell membranes also affects hemolyticactivity of the protein. This substitution, His₃₆₇→Arg, completelyinhibits hemolytic activity. This is a quite unpredictable finding inthat His₃₆₇→Arg therefore shows a greater inhibition of this propertythan the substitutions made within the 11 amino acid region thought tobe responsible for hemolytic activity. Mutations in the CRP-like domainswere tested for ability to activate complement. For Trp₃₇₉→Phe,Tyr₃₈₄→Phe, Asp₃₈₅→Asn and Trp₃₉₇→Phe, complement activation was reducedby 20%, 70%, 100% and 15%, respectively. The other mutations in theCRP-like domains shown in FIG. 4 do not reduce complement activation.

Importantly, the above mutations which affect either hemolytic activityor complement activation do not impair the immunogenicity of theproteins, compared with native or wild-type pneumolysin.

Thus although His₃₆₇→Arg is the preferred mutation to reduce thehemolytic activity, a combination of two or more mutants effectingreduced hemolytic activity can also achieve a very high level ofreduction in hemolytic activity. Similarly Asp₃₈₅→Asn is the preferredmutation to achieve reduced complement activation, however a combinationof two or more other mutants that reduce the activity to a lesser degreecan also be used.

In a preferred embodiment the pneumolysin derivative for use in thevaccine would contain a combination of certain of the above mutationssuch that the protein is unable to activate complement in addition tohaving zero hemolytic activity. Examples of such combination are:

His₃₆₇→Arg+Asp₃₈₅→Asn;  1)

His₃₆₇→Arg+Asp₃₈₅→Asn+either Cys₄₂₈→Gly or Trp₄₃₃→Phe;  2)

 Asp₃₈₅→Asn+Cys₄₂₈→Gly+Trp₄₃₃→Phe.  3)

These then are some preferred combinations, however it is to beunderstood that other combinations of mutations can be used to make upthe altered pneumolysin for use in a vaccine. Further the alteredpneumolysin may comprise any one of the individual mutations withsufficiently reduced activity.

High level expression of the altered pneumolysin from DNA encoding thealtered pneumolysin can be achieved by using any one of a number ofconventional techniques including the expression in a prokaryotic hostwith the DNA cloned appropriately within any one of the many expressionvectors currently available, or cloned appropriately within the hostchromosome; expression in a eukaryotic host with the DNA clonedappropriately either within an expression vector or cloned within thehost chromosome; or within an in vitro expression system such as maycomprise purified components necessary for expression of alteredpneumolysin.

To achieve high level expression of the mutated pneumolysin gene, it hasbeen cloned into the vector pKK233-2 for expression within Escherichiacoli or other like prokaryote. This vector included ampicillin andtetracycline resistance genes, the trc promoter (which can be regulatedby IPTG [isopropyl-B-D-thiogalactopyranosi]), and a lac Z ribosomebinding site adjacent to an ATG initiation codon incorporating an NcoIrestriction site. Immediately downstream from the initiation codon thereare restriction sites for PstI and HindIII, followed by a strong T₁ T₂transcription terminator. Prior to insertion into pKK233-2, a NcoIrestriction site was constructed at the 5′ end of the pneumolysin codingsequence (at the initiation codon) by oligonucleotide-directedmutagenesis, as shown in FIG. 2. This enabled the proximal end of thealtered pneumolysin gene to be cloned into the NcoI site of pKK233-2; aHindIII site approximately 80 bases downstream from the pneumolysintermination codon was used to splice the distal end of the altered geneinto the compatible site in pKK233-2. The mutant pneumolysin derivativecould however, be cloned into any one of a number of high expressionvector systems.

The mutant pneumolysin is prepared as follows: E. coli cells harboringthe above recombinant plasmid are first grown in 9 liter cultures inLuria Bertani (or any other appropriate) medium, supplemented with theappropriate antibiotic, at 37° C., with aeration. When the culturereaches the late logarithmic phase of growth, IPTG is added to a finalconcentration of 20 μM (to induce expression of the altered pneumolysingene) and incubation is continued for a further 2 to 3 hrs.

Cells are then harvested by centrifugation or ultrafiltration and lysedby treatment with EDTA and lysozyme, followed by sonication, or bydisruption in a French pressure cell. Cell debris is removed bycentrifugation and the extract is then dialyzed extensively against 10mM sodium phosphate (pH 7.0). The material is then loaded onto a columnof DEAE-cellulose and eluted with a linear gradient of 10-250 mM sodiumphosphate (pH 7.0). Fractions containing peak levels of the pneumolysinderivative are pooled, concentrated by ultrafiltration and loaded onto acolumn of Sephacryl S-200. This column is developed in 50 mM sodiumphosphate (pH 7.0) and again fractions with high levels of pneumolysinderivative are pooled, concentrated by ultrafiltration and stored in 50%glycerol at −15° C. The final product is greater than 95% pure, asjudged by SDS-polyacrylamide gel electrophoresis. Hydrophobicinteraction chromatography on Phenyl-Sepharose is an alternativepurification which could also be used.

However it is to be understood that this is only one method ofpurification of the altered pneumolysin, and other, alternative methods(including High Pressure Liquid Chromatography) may be employed. Thispurified altered pneumolysin can then be administered as a vaccine atappropriate levels, either by itself or in combination with otherantigens. In one form, the pneumolysin may be conjugated withpolysaccharide derived from any one or more of the variety ofpneumococcal strains described above.

The mutant pneumolysin can be conjugated to the various serotypes ofpolysaccharide by a range of methods. The first involves preparation ofan activated polysaccharide by treating pure polysaccharide (availablecommercially) with cyanogen-bromide and adipicacid dihydrazide (ADH).The ADH-polysaccharide is then combined with the mutant pneumolysin inthe presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl.Conjugated material is separated from the reactants by chromatographythrough Sepharose CL-4B.

Alternatively, the polysaccharide-mutant pneumolysin conjugates can beprepared using bifunctional reagents such asN-succinimidyl-6(4′azido-2′-nitrophenylamino)hexanoate(SANPAH). Purepolysaccharide dissolved in phosphate buffered saline, is reacted withSANPAH in the presence of a strong white light source. Unreacted SANPAHis then separated from activated polysaccharide by chromatography onSephadex G-50. Activated polysaccharide is then conjugated to the mutantpneumolysin in 0.2M borate buffer (pH 8.5). Any excess reactive groupsare then blocked with lysine, and the polysaccharide-protein conjugateis separated from the other reactants by chromatography on SepharoseCL-4B. Conjugates could also be prepared by reductive amination withcyanoborohydride.

Alternatively another protein, such as inactivated tetanus toxin, can beconjugated with the desired polysaccharides and altered pneumolysin canbe added to the vaccine in an unconjugated form. This then describes thebest method of performing the invention, however, it is to be understoodthat the invention is not limited thereto.

DETAILED DESCRIPTION OF THE INVENTION

Cloning of the Pneumolysin Gene

The cloning and DNA sequencing of the pneumolysin gene is described inthe paper by Walker et al., 1987, Infection and Immunity, 55:1184-1189). Chromosomal DNA from Streptococcus pneumoniae strain D39(NCTC 7466) was partially restricted with EcoRl and fragments werecloned into bacteriophage λ gt10. Plaques were overlaid with sheep bloodagar and hemolytic recombinants selected. The insert DNA from ahemolytic phage was sub-cloned into plasmid by digestion of therecombinant with EcoRl. The lytic product was shown to be pneumolysin asit was completely inhibited by cholesterol and reacted with antiserumraised to the related toxin Streptolysin O. The gene and its flankingDNA were sequenced following shotgun cloning into the sequencing vectorM31mp18.

Methods to Prepare Mutants of Pneumolysin

Mutant versions of the pneumolysin gene were constructed by severaldifferent methods.

Random Mutagenesis

The pneumolysin gene cloned into M13mp18 was used as a template forbase-specific misincorporation mutagenesis using a previously reportedtechnique, that allows single base-specific substitutions throughoutgenes (Lehtovarra et al., 1988, Protein Engineering, 2: 63-68).

Primary Screening Mutants for Reduced Hemolytic Activity

A method was developed to screen the library for clones with reduced orno hemolytic activity. Plaques obtained from the random mutagenesisprocedure were picked, using sterile Pasteur pipettes, into 96-wellmicrotiter plates containing 100 μl TES buffer (10 mM Tris HCl pH 8, 1mM EDTA, 50 mM NACl) in each well. Plates were stored at 4° C. for atleast 4 hours to produce a supernatant containing infectious phageparticles. An overnight culture of JM101 (Stratagene, La Jolla, Calif.)was diluted 1:100 in fresh LB medium and 150 μl of this solution wasadded to each well of a microtiter plate. Phage supernatants weretransferred to each well using a 48-spike replicator tool. Themicrotiter plates were incubated overnight at 37° C. without shaking.After overnight growth, a pellet was visible at the bottom of each well.A drop of chloroform was added to each well using the replicator tool.Chloroform addition was repeated twice to ensure lysis of phage-infectedcells, and the plates were left at room temperature for 15 mins. Using amultichannel pipette, 50 μl of supernatant from chloroform-lysed cellswas transferred to a fresh microtiter plate, and 50 μl of 2% sheep redblood cells in PBS was added. Plates were then incubated at 37° C. andexamined at various times by eye, to determine the extent of hemolysisin each well. The individual clones were ranked for their ability tolyse the red blood cell solution and those displaying least apparenthemolytic activity were selected for further analysis. This screenidentified a number of candidates with reduced or no hemolytic activity.The absence or reduction in hemolysis observed might have been due to asingle amino acid substitution, as expected. Alteratively, there mighthave been reduced expression of pneumolysin or poor infectivity ofphage. A second screen was therefore developed to eliminate clones thatdid not produce significant amounts of toxin.

Secondary Screening of Clones

All non-hemolytic clones selected by the primary screen were plaquepurified using JM101 as a host strain. JM101, infected with seriallydiluted phage from microtiter wells, was plated out at several dilutionsin soft-top media. Individual plaques were then picked from theappropriate plates that contained well separated plaques. This stepdetermined whether viable phage were present in the microtiter platewell. Four individual plaques were picked for each clone of interest andamplified in JM101. The harvested cells were sonicated and assayed forhemolytic activity. If all, four clones were non-hemolytic, a dot-blotprocedure was used to assay for toxin presence in sonicated extracts.Briefly, 5 μl of sonicated cell extracts were spotted onto anitrocellulose membrane and blocked overnight in 3% skimmed milk inPBS-Tween 20 (PBS-T). Filters were washed in PBS-T, then incubated for90 mins. with a 1:1000 dilution of rabbit anti-pneumolysin antiserum.Filters were thoroughly washed in PBS-T and incubated for 60 mins. withan IgG-peroxidase conjugate diluted 1:2000 in PBS-T. Filters were washedprior to detection by Enhanced Chemiluminescence (ECL), using methodsrecommended by the manufacturers (Amersham, UK). Clones that did notproduce toxin were eliminated from further studies. Non-hemolytic clonesthat produced toxin were analyzed by Western blotting, using ECLdetection as described above, to ensure a full-length toxin wasexpressed.

Clones with reduced hemolytic activity were also plaque purified andamplified in JM101. Two-fold serial dilutions of sonicated extracts inPBS were made in microtiter plates. JM101, infected with phage thatexpress wild-type pneumolysin, was used as a standard. An equal volumeof 2% sheep red blood cells was added to assess the hemolytic activityin a semi-quantitative manner. Single stranded DNA of clones with lessthan wild-type hemolytic activity was prepared and sequenced. Thismethod has been published by us (Hill et al., 1994, Infection andImmunity, 62: 757-758).

Site-directed Mutagenesis (SDM)

Site directed mutagenesis was done using the method of Kunkel et al.,Methods in Enzymology, 154: 367-382). M13 phage carrying the pneumolysingene was passaged through E. coli strain RZ1032 which causes theincorporation of uracil in place of thymidine at some positions in theDNA sequence. Mutant oligonucleotides were kinased and annealed to thistemplate. Following extension and ligation of the second strand using T4DNA ligase and DNA polymerase the mixture was used to transform E. colistrain JM101. Parental wild-type sequences, which contain uracil, aredegraded in this strain and the newly synthesized mutagenic strand onlyreplicates. Mutants are then selected by sequencing of DNA fromresultant plaques. Mutant genes were sequenced in their entirety andsubcloned into plasmids for protein expression.

PCR Mutagenesis (1)

Mutants were constructed by splice-extension overlap PCR (Higuchi etal., 1985, Nucleic Acid Research, 15:7351). This process uses the factthat sequences added to the 5′ end of a PCR primer become incorporatedinto the product molecule. PCR was used to modify the ends of twofragments of the pneumolysin gene. The modified sequences were designedso that they overlap and were then used in an overlap extension reactionto incorporate the mutation into the appropriate position in thepneumolysin gene.

PCR Mutagenesis (2)

Mutants were constructed by standard PCR amplification of thepneumolysin gene using a mutagenic 3′ oligonucleotide.

Preparation of a High Expression Vector Encoding Mutant Pneumolysin

The gene encoding for mutant pneumolysin is inserted immediatelydownstream from the T7 promoter of the pGEMEX-1 vector utilizing a NdeIsite. Therefore a NdeI site was introduced in the initiation codon ofthe pneumolysin gene by oligonucleotide-directed mutagenesis. For thepGEMEX-1 system, a NdeI site from the vector at position 3251 wasremoved by deleting a 860 bp SppI fragment (position 2884-3744). Thisremoves the f1 ori region, which is not needed in this construct. A NdeIsite is left at position 904 in the initiation codon of the T7 gen.Digestion of the vector with NdeI and SphI removed a further 883 bp ofDNA encoding the T7 gene 10 and most of the polylinker region. Thisenabled insertion of the pneumolysin coding sequence as a 1.5 kbNdeI/SphI fragment into pGEMEX-1. (FIG. 5 shows the physical map of thepGEMEX-1 vector encoding for mutant pneumolysin.)

Expression of Mutated Pneumolysin in E. coli

The constructed plasmid was introduced by transformation of the strainE. coli JM109. E. coli cells bearing plasmids were selected on LB mediumcontaining 50 μg/ml ampicillin. Subsequently the clones were screenedfor the presence of pneumolysin sequence by dot-blot hybridization topneumolysin gene probes, and clones expressing intact mutant pneumolysin(pneumolysoid) were detected by Western blot analysis. analysis. PlasmidDNA was also extracted and subjected to restriction analysis to confirmthe physical structure.

The transformed cells were then inoculated in 2 bottles (30 ml) eachcontaining 10 ml of LB medium with 50 μg/ml ampicillin and subsequentlygrown at 37° C. for 3-4 hrs. At the end of this period the 10 ml of eachculture were added to 500 ml of fresh LB medium in one liter flasks andwere kept under agitation for 3-4 hrs. at 37° C. These cultures werethen used to inoculate 2.5 liters of LB medium with ampicillin (50μg/ml) supplemented with 10 ml of 20% glucose in a New Brunswick BiofloII fermenter (5 liter capacity vessel). The cells were grown overnightat 37° C. with maximum aeration, agitation at 500 rpm. pH was maintainedat 6.8-7.0 with HCl or NH₄OH. Next morning one liter LB medium with 50μg/ml ampicillin, 20 ml 20% (w/v) glucose as well as 200 mgisopropyl-β-thiogalactoside were added. The culture was continued foranother 2 h. The cultures were centrifuged and the cells separated andresuspended in 70 ml of 10 mM sodiumphosphate pH 7.0.

Purification of Mutated Pneumolysin

The said cell suspension was then lysed by passage through a Frenchpressure cell at 15,000 psi. The cell lysate was centrifuged at 35,000×gfor 20 mins. at 4° C. The supernatant was separated and centrifuged at150,000×g for 60 mins. at 4° C. The supernatant containing solublepneumolysoid was used for purification.

The crude supernatant was loaded onto a DEAE Sepharose CL-6B column (5cm×60 cm) at 4° C. The column was eluted with a 2 liter linear gradientof 10-250 mM sodiumphosphate pH 7.0 applied at a flow rate of 100 ml/h.The column effluent was collected in fractions of 20 ml. Fractionscontaining detoxified pneumolysin were identified by a sandwich ELISAusing rabbit anti-pneumolysin serum and mouse anti-pneumolysinmonoclonal or a dot-blot immunoassay using rabbit anti-pneumolysin. Peakfractions containing pneumolysoid were pooled and concentrated byultrafiltration using an Amicon 250 ml capacity stirred cell with anYM10 (10,000 MW cut off) membrane. The partially purified pneumolysoidis then loaded onto a Sephacryl S200 HR column (2.6×100 cm). The columnwas equilibrated and eluted with 50 mM sodiumphosphate pH 7.0 at a flowrate of 30 ml/h. Fractions of 10 ml were collected and assayed asbefore. Peak fractions containing detoxified pneumolysin wereconcentrated by ultrafiltration up to a maximum of 5 mg/ml. The yieldwas 30-50 mg with a purity estimated with SDS-PAGE to be 90-95% Thepurified pneumolysoid is then supplemented to 50% glycerol and stored at−15° C.

Various samples were taken during purification of pneumolysoid andsubjected to electrophoresis. FIG. 6 shows the results of theelectrophoresis, in which Lane 1 contains molecular weight markers 97.4,66.2, 45.0, 31.0 and 21.0 kDa from top to bottom, Lane 2 contains crudeE. coli lysate, Lane 3 contains post DEAE Sepharose CL-6B, and Lane 4contains post Sephacryl S200HR. Ten μl of samples taken duringpurification had added 0.05% bromophenol blue, 5% β-mercaptoethanol, 10%glycerol, they were brought to boiling point for five mins. and thenloaded onto a 12.5% polyacrylamide gel. The proteins were then subjectedto electrophoresis at 200 V for 3 hrs. and the gel was colored anddecolored as reported by Laemli (Nature 227, 680-685).

In vitro Characterization of Mutant Pneumolysin

Hemolytic Activity

Doubling dilutions (50 μl) of toxin solution are added to an equalvolume of sheep red blood cells. That dilution which causes lysis of 50%of the cells is taken as the end point. The reciprocal of this dilutionis taken as the number of hemolytic units. This value is normalized forthe concentration of the toxin and is expressed as hemolytic units permilligram of protein.

Complement Activation

10 μg of toxin was incubated in normal human serum at 37° C. for 30mins. Complement activation was measured by using two-dimensionalimmunoelectrophoresis to visualize the C3b released (Mitchell et al.,1989, Biochim. Biophys. Acta, 1007:67-72).

Cell Binding

Dilutions of the toxin, from 260 to 3 ng/ml, were made in 3 ml of Hanksbuffered salts solution (HBSS) containing 0.2% (v/v) sheep erythrocytesand incubated in an ice-water bath for 30 mins. The cells were washedthree times in ice-cold HBSS, then lysed in water. The membranes wereharvested and washed twice in water by centrifugation at 13,000 rpm in amicrofuge and then resuspended in sodium dodecylsulphate-polyacrylamidegel electrophoresis (SDS-PA-GE) loading buffer.SDS-PAGE was done and the proteins were transferred to nitrocellulosemembranes. The membranes were incubated in 5% (w/v) skimmed milk powderin PBS for 60 mins., washed in PBS and incubated with rabbitanti-pneumolysin antiserum, diluted 1/1000 in PBS, for 90 mins. Themembranes were washed, incubated for 60 mins. in 1/2000 goat anti-rabbitantiserum and then extensively washed in PBS. The proteins recognized bythe anti-pneumolysin antiserum were visualized using EnhancedChemiluminescence reagents (Amersham) following the manufacturer'sinstructions. The results were quantified using an LKB Ultrascan XLdensitometer.

Data on the in vitro biological activity of mutants of pneumolysin arepresented in Table 1.

Thus, the data in Table 1 illustrates that the present inventionprovides substantially reduced hemolytic activity and reduced complementbonding activity as compared to wild-type pneumolysins.

In vivo Characterization of Mutant Pneumolysin (Trp₄₃₃→Phe)

Preparation of Anti-pneumolysin Serum

New Zealand white rabbits were injected with 10 μg of purifiedpneumolysin in Freund's complete adjuvant intramuscularly three times atthree week intervals. Three weeks after the final intramuscularinjection rabbits were boosted with 10 μg of pneumolysin intravenouslyand were bled from the ear vein one week later.

Analysis of Serum Response to Immunization with Detoxified MutantPneumolysin (Trp₁₃₃→Phe

Female MFl mice (Harlan, Olac Ltd.) weighing 30-35 g were injectedintraperitoneally with 20 μg of purified pneumolysoid. One group of micewere immunized with 0.1 ml phosphate buffered saline, 50% v/v glycerol,emulsified with 0.1 ml Freund's complete adjuvant (Sigma Chemical Co.Poole, UK). At 10-day intervals the mice were given two additionalinjections of 20 μg of purified mutant pneumolysin in PBS, 50% v/vglycerol, emulsified with Freund's incomplete adjuvant (Sigma, Poole,UK). Another group of mice was injected intraperitoneally with 20 μg ofpurified pneumolysin adsorbed to aluminum phosphate. At 10-day intervalsthe mice were given two additional injections. As a control for each ofthese two groups of mice, groups of mice were immunized according to thesame protocol with adjuvant only. Blood samples were taken from the tailvein before each injection and before challenge. Sera were stored at−20° C. until use. Sera from mice in which aluminum phosphate was usedas the adjuvant were assayed for the presence of anti-pneumolysinantibodies in ELISA. The results expressed as average from 10 animalstested individually are reported in FIG. 7. In FIG. 7, the solid linerepresents the mice vaccinated with the pneumolysin in alum adjuvant(aluminum phosphate), the dotted line represents the pneumolysoid inFreunds adjuvant, and the dashed line represents the control using PBSadjuvant only. Each point represents the arithmetic mean of ten mice,and the bars represent the standard error of the mean.

The sera from mice immunized with toxoid and Freunds adjuvant wereanalyzed from their isotypes profile. The results (FIG. 8) showed thatthe major isotypes present were IgG1 and IgG2. In FIG. 8, each columnrepresents the arithmetic mean of ten mice, and the bars represent thestandard error of the mean.

Intranasal Challenge with Various Pneumococcal Serotypes afterImmunization with Mutant Pneumolysin

Preparation of a Standard Pneumococcal Inoculum

Pneumococcal strains (supplied by RIVM, clinical isolates from LeicesterRoyal Infirmary or purchased from NCTC) were inoculated into brain heartinfusion broth (BHI, Oxoid) and grown up overnight. 100 μl of theovernight culture were injected intraperitoneally into an MFI mouse.Organisms were recovered from the mouse by plating a blood sample takenfrom the tail vein 24 hrs. later onto blood agar base (BAB, Oxoid)containing 5% (v/v) horse blood. Plates were incubated overnight at 37°C. Four-five colonies were then inoculated in bottles each containing 10ml of brain-heart infusion broth (BHI) and were grown overnight at 37°C. Next morning the culture was centrifuged, the pellet was separatedand resuspended in 1 ml of BHI supplemented with 17% (v/v) fetal calfserum (FCS). The bacterial suspension was diluted with fresh medium upto OD of 0.7 was reached. This culture was then incubated at 37° C. for4-5 hrs. The number of viable cells were estimated by plating thebacteria on BAB+5% horse blood in triplicate and incubating the platesovernight at 37° C. The remainder of the culture was frozen at −20° C.Next day the cells were thawed and diluted with BHI+FCS to aconcentration of 2×10₇ cfu per ml aliquoted in 1 ml portions and storedat −70° C. until use. When required, the suspension was thawed slowly atroom temperature and bacteria were harvested by centrifugation beforeresuspension to the required concentration in sterile PBS.

Intranasal Challenge

Female MFI mice, weighing approximately 30-35 g, were immunized withmutant pneumolysin as described above using aluminum phosphate as theadjuvant.

One month after the third injection, mice were lightly anesthetized with80 μl of 1 mg/ml Hypnorm (Janssen) given intraperitoneally. Twentyminutes after administration of the anaesthetic, mice were challengedwith 50 μl of PBS containing the number of colony forming units of S.pneumoniae strains (see Table 2), which caused the control mice tobecome moribund in approximately 3 days, administered in the nostrils.Challenged mice were kept warm until they had recovered consciousness(2-3 hrs.) The mice were monitored for visible clinical symptoms for 14days, at which the experiment was ended. Mice that were alive at thispoint were considered to have survived the pneumococcal challenge. Micethat became moribund was recorded and the animal was killed humanely.The survival time of each mouse is given in Table 2. The results givenin this table indicate that a significant increase in the survival timesof immunized mice was seen in each case except for type 3 strain GBO5.

Intraperitoneal Challenge with Various Pneumococcal Serotypes of MiceRepeatedly Immunized with Mutant Pneumolysin

Preparation of a Standard Pneumococcal Inoculum

Pneumococcal strains stored at −80° C. in serum broth (meat extractbroth+10% horse serum) was used to inoculate a blood agar plate. Plateswere incubated overnight at 37° C. After that one loop of cells wassuspended in 3 ml of serum broth. The culture was incubated at 37° C.until an OD₆₀₀ of 0.2 corresponding with 1.0×10⁸ cfu/ml. The culture wasdiluted in serum broth, as appropriate immediately prior tointraperitoneal challenge.

Intraperitoneal Challenge

Male and female Quackenbush strain (Q/S) mice, 6-8 weeks old, wereinjected subcutaneously with 0.1 ml volumes of a 20% suspension of alumadjuvant (Imjcet Alum, Pierce, Rockford, Ill.) in PBS, containing 20 μgof mutant pneumolysin. Mice in control groups received 0.1 ml of asuspension of 20% alum and PBS. At 14 day intervals mice were givenadditional injections. Two weeks after the third injection mice werechallenged intraperitoneally with a 0.1 ml dose of pneumococcicalculated to represent approximately 20 times the 50% lethal dose foreach strain. The time of death of mice over the subsequent 14 day periodwas recorded. The experiment was ended after 14 days and mice alive atthis time were recorded as survivors.

Groups of 15 immunized and non-immunized Q/S mice were challengedintraperitoneally with the dose of each strain of pneumococcus shown inTable 3. The survival times of the mice was recorded and is presented inTable 3. The results indicate that the survival times of the miceimmunized with mutant pneumolysin were significantly increased withrespect to those of the control mice with each of the eight challengestrains of pneumococcus.

Parenteral Vaccine Preparation

5-50 g of the mutant pneumolysin is mixed with an aluminum adjuvant suchas aluminum phosphate, to produce a vaccine in a form appropriate forincorporation into a parenteral administration dosage form.

The vaccine of the invention may be prepared as a pharmaceuticalcomposition containing an immunoprotective, non toxic amount of theprotein of the invention in a non toxic and sterile pharmaceuticallyacceptable carrier. The mode of administration of the vaccine of theinvention may be any suitable route which delivers protection toinfection with virulent pneumococci. Where the vaccine is administeredparenterally, via the intramuscular or deep subcutaneous route, theprotein can be optionally admixed or absorbed with any conventionaladjuvant to attract or to enhance the immune response. Such adjuvantsinclude but are not restricted to aluminum hydroxide, aluminumphosphate, muramyl dipeptide, bacterial lipopolysaccharides andderivatives and purified saponins from Quil A. The protein can also bepresented to the immune system within microparticles such as liposomesor ISCOMs. A vaccine formulation containing the protein of the inventionmay be designed for oral or intranasal ingestion.

The therapeutically effective and non toxic dose of the vaccine can bedetermined by skill in the art. However the specific dose for any personwill depend upon a variety of factors including age, general health,diet of the patient, time and route of administration, synergisticeffects with other drugs being administered and whether the vaccine isadministered repeatedly. If necessary the vaccine will be administeredrepeatedly with one to three month intervals between each dose and withan optional booster dose later in time.

TABLE 1 Data on the in vitro biological activity of mutants ofpneumolysin. haemolytic complement cell Method used pneumolysin activityactivity binding to construct mutant (%) (%) (%) mutant Arg31 -->Cys 75ND ND RANDOM Leu75 -->Phe 100 ND ND RANDOM Val127 -->Gly 75 ND ND RANDOMHis156 -->Tyr 0.1 ND ND RANDOM His367 -->Arg 0.1 ND ND SDM Asp385 -->Asn100 <1 100 SDM His386 -->Arg 10 ND ND PCR(1) Glu390 -->Asp Cys428 -->Ala100 100 100 SDM Cys428 -->Gly 1 100 100 SDM Cys428 -->Ser 10 100 100 SDMAla432 -->Val 100 ND ND RANDOM Trp433 -->Arg 0.01 ND ND RANDOM Trp433-->Phe 0.1 100 100 SDM Glu434 -->Gln 20 100 100 SDM Glu434 -->Asp 50 100100 SDM Trp435 -->Phe 10 100 100 SDM Trp436 -->Phe 100 100 100 SDMTrp436 -->Arg 50 ND ND RANDOM Pro462 -->Ser 25 ND  10 PCR(2) Asp385-->Asn 10 ND ND Cloning Cys428 -->Gly His156 -->Tyr 0.1 ND ND CloningAsp385 -->Asn His156 -->Tyr <0.001 ND ND Asp385 -->Asn Trp433 -->PheTrp433 -->Phe 0.5 ND ND SDM Asp385 -->Asn His367 -->Arg 0.1 ND NDCloning Asp385 -->Asn His367 -->Arg 0 ND ND Cloning Asp385 -->Asn Trp433-->Phe ND = Not done Note: Data are presented as compared to nativepneumolysine (%).

TABLE 2 Protection of mice against intraperitoneal challenge withvarious S. pneumoniae strains elicited by immunization with PdB.Survival Time (days) % Survival Con- Im- Con- Im- Type HU^(a) Dose^(b)trol mune p^(c) trol mune p^(d)  1 621 1 × 10⁷ 5.1 >14 <0.001 13 60<0.005   3 335 1 × 10⁵ 3.0 3.8 <0.025 13 0 NS  4 585 4 × 10³ 1.8 >14<0.003 7 73 <0.0005  5 693 2 × 10⁸ 5.5 9.3 <0.005 15 36 NS  6 424 1 ×10⁷ 3.9 >14 <0.001 7 67 <0.005  7F 221 2 × 10⁷ 1.8 7.9 <0.05 20 33 NS  823 4 × 10³ 1.2 1.5 <0.003 0 7 NS 18C 330 1 × 10⁵ 1.8 2.3 <0.03 0 14 NS^(a)Pneumolysin production expressed as HU per ml culture at A₆₀₀ = 1.0.^(b)Challenge dose in number of colony forming units ^(c)Significance ofdifference, Mann-Whitney U-test ^(d)Significance of difference, χ² test

TABLE 3 Protection of mice against intranasal challenge with various S.pneumoniae strains elicited by immunization with PdB. Survival Time(days) % Survival HU/ Con- Im- Con- Im- Type ml^(a) Dose^(b) trol munep^(c) trol mune p^(d)  1N 160 4.0 × 10⁶ 3.0 3.9 <0.05 10 40 <0.01  2 6401.0 × 10⁶ 3.2 >14.0 <0.01 6 72 <0.01  3 320 1.0 × 10⁶ 2.8 4.0 <0.01 1033 <0.01  7F 140 2.0 × 10⁶ 2.9 >14.0 <0.01 0 70 <0.01 18C 260 1.0 × 10⁷3.2 >14.0 <0.01 5 85 <0.01 ^(a)One hemolytic unit (HU) is defined as theamount of pneumolysin that causes 50% hemolysis of 1% sheep erythrocyteswhen incubated at 37° C. for 30 min. ^(b)Challenge dose in number ofcolony forming units ^(c)Significance of difference, Mann-Whitney U-test^(d)Significance of difference, χ² test

6 1415 base pairs nucleic acid single linear cDNA CDS 3..1415 1 AG ATGGCA AAT AAA GCA GTA AAT GAC TTT ATA CTA GCT ATG AAT TAC 47 Met Ala AsnLys Ala Val Asn Asp Phe Ile Leu Ala Met Asn Tyr 1 5 10 15 GAT AAA AAGAAA CTC TTG ACC CAT CAG GGA GAA AGT ATT GAA AAT CGT 95 Asp Lys Lys LysLeu Leu Thr His Gln Gly Glu Ser Ile Glu Asn Arg 20 25 30 TTC ATC AAA GAGGGT AAT CAG CTA CCC GAT GAG TTT GTT GTT ATC GAA 143 Phe Ile Lys Glu GlyAsn Gln Leu Pro Asp Glu Phe Val Val Ile Glu 35 40 45 AGA AAG AAG CGG AGCTTG TCG ACA AAT ACA AGT GAT ATT TCT GTA ACA 191 Arg Lys Lys Arg Ser LeuSer Thr Asn Thr Ser Asp Ile Ser Val Thr 50 55 60 GCT ACC AAC GAC AGT CGCCTC TAT CCT GGA GCA CTT CTC GTA GTG GAT 239 Ala Thr Asn Asp Ser Arg LeuTyr Pro Gly Ala Leu Leu Val Val Asp 65 70 75 GAG ACC TTG TTA GAG AAT AATCCC ACT CTT CTT GCG GTT GAT CGT GCT 287 Glu Thr Leu Leu Glu Asn Asn ProThr Leu Leu Ala Val Asp Arg Ala 80 85 90 95 CCG ATG ACT TAT AGT ATT GATTTG CCT GGT TTG GCA AGT AGC GAT AGC 335 Pro Met Thr Tyr Ser Ile Asp LeuPro Gly Leu Ala Ser Ser Asp Ser 100 105 110 TTT CTC CAA GTG GAA GAC CCCAGC AAT TCA AGT GTT CGC GGA GCG GTA 383 Phe Leu Gln Val Glu Asp Pro SerAsn Ser Ser Val Arg Gly Ala Val 115 120 125 AAC GAT TTG TTG GCT AAG TGGCAT CAA GAT TAT GGT CAG GTC AAT AAT 431 Asn Asp Leu Leu Ala Lys Trp HisGln Asp Tyr Gly Gln Val Asn Asn 130 135 140 GTC CCA GCT AGA ATG CAG TATGAA AAA ATA ACG GCT CAC AGC ATG GAA 479 Val Pro Ala Arg Met Gln Tyr GluLys Ile Thr Ala His Ser Met Glu 145 150 155 CAA CTC AAG GTC AAG TTT GGTTCT GAC TTT GAA AAG ACA GGG AAT TCT 527 Gln Leu Lys Val Lys Phe Gly SerAsp Phe Glu Lys Thr Gly Asn Ser 160 165 170 175 CTT GAT ATT GAT TTT AACTCT GTC CAT TCA GGT GAA AAG CAG ATT CAG 575 Leu Asp Ile Asp Phe Asn SerVal His Ser Gly Glu Lys Gln Ile Gln 180 185 190 ATT GTT AAT TTT AAG CAGATT TAT TAT ACA GTC AGC GTA GAC GCT GTT 623 Ile Val Asn Phe Lys Gln IleTyr Tyr Thr Val Ser Val Asp Ala Val 195 200 205 AAA AAT CCA GGA GAT GTGTTT CAA GAT ACT GTA ACG GTA GAG GAT TTA 671 Lys Asn Pro Gly Asp Val PheGln Asp Thr Val Thr Val Glu Asp Leu 210 215 220 AAA CAG AGA GGA ATT TCTGCA GAG CGT CCT TTG GTC TAT ATT TCG AGT 719 Lys Gln Arg Gly Ile Ser AlaGlu Arg Pro Leu Val Tyr Ile Ser Ser 225 230 235 GTT GCT TAT GGG CGC CAAGTC TAT CTC AAG TTG GAA ACC ACG AGT AAG 767 Val Ala Tyr Gly Arg Gln ValTyr Leu Lys Leu Glu Thr Thr Ser Lys 240 245 250 255 AGT GAT GAA GTA GAGGCT GCT TTT GAA GCT TTG ATA AAA GGA GTC AAG 815 Ser Asp Glu Val Glu AlaAla Phe Glu Ala Leu Ile Lys Gly Val Lys 260 265 270 GTA GCT CCT CAG ACAGAG TGG AAG CAG ATT TTG GAC AAT ACA GAA GTG 863 Val Ala Pro Gln Thr GluTrp Lys Gln Ile Leu Asp Asn Thr Glu Val 275 280 285 AAG GCG GTT ATT TTAGGG GGC GAC CCA AGT TCG GGT GCC CGA GTT GTA 911 Lys Ala Val Ile Leu GlyGly Asp Pro Ser Ser Gly Ala Arg Val Val 290 295 300 ACA GGC AAG GTG GATATG GTA GAG GAC TTG ATT CAA GAA GGC AGT CGC 959 Thr Gly Lys Val Asp MetVal Glu Asp Leu Ile Gln Glu Gly Ser Arg 305 310 315 TTT ACA GCA GAT CATCCA GGC TTG CCG ATT TCC TAT ACA ACT TCT TTT 1007 Phe Thr Ala Asp His ProGly Leu Pro Ile Ser Tyr Thr Thr Ser Phe 320 325 330 335 TTA CGT GAC AATGTA GTT GCG ACC TTT CAA AAC AGT ACA GAC TAT GTT 1055 Leu Arg Asp Asn ValVal Ala Thr Phe Gln Asn Ser Thr Asp Tyr Val 340 345 350 GAG ACT AAG GTTACA GCT TAC AGA AAC GGA GAT TTA CTG CTG GAT CAT 1103 Glu Thr Lys Val ThrAla Tyr Arg Asn Gly Asp Leu Leu Leu Asp His 355 360 365 AGT GGT GCC TATGTT GCC CAA TAT TAT ATT ACT TGG GAT GAA TTA TCC 1151 Ser Gly Ala Tyr ValAla Gln Tyr Tyr Ile Thr Trp Asp Glu Leu Ser 370 375 380 TAT GAT CAT CAAGGT AAG GAA GTC TTG ACT CCT AAG GCT TGG GAC AGA 1199 Tyr Asp His Gln GlyLys Glu Val Leu Thr Pro Lys Ala Trp Asp Arg 385 390 395 AAT GGG CAG GATTTG ACG GCT CAC TTT ACC ACT AGT ATT CCT TTA AAA 1247 Asn Gly Gln Asp LeuThr Ala His Phe Thr Thr Ser Ile Pro Leu Lys 400 405 410 415 GGG AAT GTTCGT AAT CTC TCT GTC AAA ATT AGA GAG TGT ACC GGG CTT 1295 Gly Asn Val ArgAsn Leu Ser Val Lys Ile Arg Glu Cys Thr Gly Leu 420 425 430 GCC TGG GAATGG TGG CGT ACG GTT TAT GAA AAA ACC GAT TTG CCA CTA 1343 Ala Trp Glu TrpTrp Arg Thr Val Tyr Glu Lys Thr Asp Leu Pro Leu 435 440 445 GTG CGT AAGCGG ACG ATT TCT ATT TGG GGA ACA ACT CTC TAT CCT CAG 1391 Val Arg Lys ArgThr Ile Ser Ile Trp Gly Thr Thr Leu Tyr Pro Gln 450 455 460 GTA GAG GATAAG GTA GAA AAT GAC 1415 Val Glu Asp Lys Val Glu Asn Asp 465 470 471amino acids amino acid linear protein 2 Met Ala Asn Lys Ala Val Asn AspPhe Ile Leu Ala Met Asn Tyr Asp 1 5 10 15 Lys Lys Lys Leu Leu Thr HisGln Gly Glu Ser Ile Glu Asn Arg Phe 20 25 30 Ile Lys Glu Gly Asn Gln LeuPro Asp Glu Phe Val Val Ile Glu Arg 35 40 45 Lys Lys Arg Ser Leu Ser ThrAsn Thr Ser Asp Ile Ser Val Thr Ala 50 55 60 Thr Asn Asp Ser Arg Leu TyrPro Gly Ala Leu Leu Val Val Asp Glu 65 70 75 80 Thr Leu Leu Glu Asn AsnPro Thr Leu Leu Ala Val Asp Arg Ala Pro 85 90 95 Met Thr Tyr Ser Ile AspLeu Pro Gly Leu Ala Ser Ser Asp Ser Phe 100 105 110 Leu Gln Val Glu AspPro Ser Asn Ser Ser Val Arg Gly Ala Val Asn 115 120 125 Asp Leu Leu AlaLys Trp His Gln Asp Tyr Gly Gln Val Asn Asn Val 130 135 140 Pro Ala ArgMet Gln Tyr Glu Lys Ile Thr Ala His Ser Met Glu Gln 145 150 155 160 LeuLys Val Lys Phe Gly Ser Asp Phe Glu Lys Thr Gly Asn Ser Leu 165 170 175Asp Ile Asp Phe Asn Ser Val His Ser Gly Glu Lys Gln Ile Gln Ile 180 185190 Val Asn Phe Lys Gln Ile Tyr Tyr Thr Val Ser Val Asp Ala Val Lys 195200 205 Asn Pro Gly Asp Val Phe Gln Asp Thr Val Thr Val Glu Asp Leu Lys210 215 220 Gln Arg Gly Ile Ser Ala Glu Arg Pro Leu Val Tyr Ile Ser SerVal 225 230 235 240 Ala Tyr Gly Arg Gln Val Tyr Leu Lys Leu Glu Thr ThrSer Lys Ser 245 250 255 Asp Glu Val Glu Ala Ala Phe Glu Ala Leu Ile LysGly Val Lys Val 260 265 270 Ala Pro Gln Thr Glu Trp Lys Gln Ile Leu AspAsn Thr Glu Val Lys 275 280 285 Ala Val Ile Leu Gly Gly Asp Pro Ser SerGly Ala Arg Val Val Thr 290 295 300 Gly Lys Val Asp Met Val Glu Asp LeuIle Gln Glu Gly Ser Arg Phe 305 310 315 320 Thr Ala Asp His Pro Gly LeuPro Ile Ser Tyr Thr Thr Ser Phe Leu 325 330 335 Arg Asp Asn Val Val AlaThr Phe Gln Asn Ser Thr Asp Tyr Val Glu 340 345 350 Thr Lys Val Thr AlaTyr Arg Asn Gly Asp Leu Leu Leu Asp His Ser 355 360 365 Gly Ala Tyr ValAla Gln Tyr Tyr Ile Thr Trp Asp Glu Leu Ser Tyr 370 375 380 Asp His GlnGly Lys Glu Val Leu Thr Pro Lys Ala Trp Asp Arg Asn 385 390 395 400 GlyGln Asp Leu Thr Ala His Phe Thr Thr Ser Ile Pro Leu Lys Gly 405 410 415Asn Val Arg Asn Leu Ser Val Lys Ile Arg Glu Cys Thr Gly Leu Ala 420 425430 Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys Thr Asp Leu Pro Leu Val 435440 445 Arg Lys Arg Thr Ile Ser Ile Trp Gly Thr Thr Leu Tyr Pro Gln Val450 455 460 Glu Asp Lys Val Glu Asn Asp 465 470 1415 base pairs nucleicacid single linear cDNA 3 CCATGGCAAA TAAAGCAGTA AATGACTTTA TACTAGCTATGAATTACGAT AAAAAGAAAC 60 TCTTGACCCA TCAGGGAGAA AGTATTGAAA ATCGTTTCATCAAAGAGGGT AATCAGCTAC 120 CCGATGAGTT TGTTGTTATC GAAAGAAAGA AGCGGAGCTTGTCGACAAAT ACAAGTGATA 180 TTTCTGTAAC AGCTACCAAC GACAGTCGCC TCTATCCTGGAGCACTTCTC GTAGTGGATG 240 AGACCTTGTT AGAGAATAAT CCCACTCTTC TTGCGGTTGATCGTGCTCCG ATGACTTATA 300 GTATTGATTT GCCTGGTTTG GCAAGTAGCG ATAGCTTTCTCCAAGTGGAA GACCCCAGCA 360 ATTCAAGTGT TCGCGGAGCG GTAAACGATT TGTTGGCTAAGTGGCATCAA GATTATGGTC 420 AGGTCAATAA TGTCCCAGCT AGAATGCAGT ATGAAAAAATAACGGCTCAC AGCATGGAAC 480 AACTCAAGGT CAAGTTTGGT TCTGACTTTG AAAAGACAGGGAATTCTCTT GATATTGATT 540 TTAACTCTGT CCATTCAGGT GAAAAGCAGA TTCAGATTGTTAATTTTAAG CAGATTTATT 600 ATACAGTCAG CGTAGACGCT GTTAAAAATC CAGGAGATGTGTTTCAAGAT ACTGTAACGG 660 TAGAGGATTT AAAACAGAGA GGAATTTCTG CAGAGCGTCCTTTGGTCTAT ATTTCGAGTG 720 TTGCTTATGG GCGCCAAGTC TATCTCAAGT TGGAAACCACGAGTAAGAGT GATGAAGTAG 780 AGGCTGCTTT TGAAGCTTTG ATAAAAGGAG TCAAGGTAGCTCCTCAGACA GAGTGGAAGC 840 AGATTTTGGA CAATACAGAA GTGAAGGCGG TTATTTTAGGGGGCGACCCA AGTTCGGGTG 900 CCCGAGTTGT AACAGGCAAG GTGGATATGG TAGAGGACTTGATTCAAGAA GGCAGTCGCT 960 TTACAGCAGA TCATCCAGGC TTGCCGATTT CCTATACAACTTCTTTTTTA CGTGACAATG 1020 TAGTTGCGAC CTTTCAAAAC AGTACAGACT ATGTTGAGACTAAGGTTACA GCTTACAGAA 1080 ACGGAGATTT ACTGCTGGAT CATAGTGGTG CCTATGTTGCCCAATATTAT ATTACTTGGG 1140 ATGAATTATC CTATGATCAT CAAGGTAAGG AAGTCTTGACTCCTAAGGCT TGGGACAGAA 1200 ATGGGCAGGA TTTGACGGCT CACTTTACCA CTAGTATTCCTTTAAAAGGG AATGTTCGTA 1260 ATCTCTCTGT CAAAATTAGA GAGTGTACCG GGCTTGCCTGGGAATGGTGG CGTACGGTTT 1320 ATGAAAAAAC CGATTTGCCA CTAGTGCGTA AGCGGACGATTTCTATTTGG GGAACAACTC 1380 TCTATCCTCA GGTAGAGGAT AAGGTAGAAA ATGAC 1415471 amino acids amino acid linear protein 4 Met Ala Asn Lys Ala Val AsnAsp Phe Ile Leu Ala Met Asn Tyr Asp 1 5 10 15 Lys Lys Lys Leu Leu ThrHis Gln Gly Glu Ser Ile Glu Asn Arg Phe 20 25 30 Ile Lys Glu Gly Asn GlnLeu Pro Asp Glu Phe Val Val Ile Glu Arg 35 40 45 Lys Lys Arg Ser Leu SerThr Asn Thr Ser Asp Ile Ser Val Thr Ala 50 55 60 Thr Asn Asp Ser Arg LeuTyr Pro Gly Ala Leu Leu Val Val Asp Glu 65 70 75 80 Thr Leu Leu Glu AsnAsn Pro Thr Leu Leu Ala Val Asp Arg Ala Pro 85 90 95 Met Thr Tyr Ser IleAsp Leu Pro Gly Leu Ala Ser Ser Asp Ser Phe 100 105 110 Leu Gln Val GluAsp Pro Ser Asn Ser Ser Val Arg Gly Ala Val Asn 115 120 125 Asp Leu LeuAla Lys Trp His Gln Asp Tyr Gly Gln Val Asn Asn Val 130 135 140 Pro AlaArg Met Gln Tyr Glu Lys Ile Thr Ala His Ser Met Glu Gln 145 150 155 160Leu Lys Val Lys Phe Gly Ser Asp Phe Glu Lys Thr Gly Asn Ser Leu 165 170175 Asp Ile Asp Phe Asn Ser Val His Ser Gly Glu Lys Gln Ile Gln Ile 180185 190 Val Asn Phe Lys Gln Ile Tyr Tyr Thr Val Ser Val Asp Ala Val Lys195 200 205 Asn Pro Gly Asp Val Phe Gln Asp Thr Val Thr Val Glu Asp LeuLys 210 215 220 Gln Arg Gly Ile Ser Ala Glu Arg Pro Leu Val Tyr Ile SerSer Val 225 230 235 240 Ala Tyr Gly Arg Gln Val Tyr Leu Lys Leu Glu ThrThr Ser Lys Ser 245 250 255 Asp Glu Val Glu Ala Ala Xaa Glu Ala Leu IleLys Gly Val Lys Val 260 265 270 Ala Pro Gln Thr Glu Xaa Lys Gln Ile LeuAsp Asn Thr Glu Val Lys 275 280 285 Ala Val Ile Leu Gly Gly Asp Pro SerSer Gly Ala Arg Val Val Thr 290 295 300 Gly Lys Val Asp Met Val Glu AspLeu Ile Gln Glu Gly Ser Arg Phe 305 310 315 320 Thr Ala Asp His Pro GlyLeu Pro Ile Ser Tyr Thr Thr Ser Phe Leu 325 330 335 Arg Asp Asn Val ValAla Thr Phe Gln Asn Ser Thr Asp Tyr Val Glu 340 345 350 Thr Lys Val ThrAla Tyr Arg Asn Gly Asp Leu Leu Leu Asp Xaa Ser 355 360 365 Gly Ala TyrVal Ala Gln Tyr Tyr Ile Thr Xaa Asp Glu Leu Ser Xaa 370 375 380 Xaa HisGln Gly Lys Glu Val Leu Thr Pro Lys Ala Xaa Asp Arg Asn 385 390 395 400Gly Gln Asp Leu Thr Ala His Phe Thr Thr Ser Ile Pro Leu Lys Gly 405 410415 Asn Val Arg Asn Leu Ser Val Lys Ile Arg Glu Xaa Thr Gly Leu Ala 420425 430 Xaa Xaa Xaa Xaa Arg Thr Val Tyr Glu Lys Thr Asp Leu Pro Leu Val435 440 445 Arg Lys Arg Thr Ile Ser Ile Trp Gly Thr Thr Leu Tyr Pro GlnVal 450 455 460 Glu Asp Lys Val Glu Asn Asp 465 470 471 amino acidsamino acid linear protein 5 Met Ala Asn Lys Ala Val Asn Asp Phe Ile LeuAla Met Asn Tyr Asp 1 5 10 15 Lys Lys Lys Leu Leu Thr His Gln Gly GluSer Ile Glu Asn Arg Phe 20 25 30 Ile Lys Glu Gly Asn Gln Leu Pro Asp GluPhe Val Val Ile Glu Arg 35 40 45 Lys Lys Arg Ser Leu Ser Thr Asn Thr SerAsp Ile Ser Val Thr Ala 50 55 60 Thr Asn Asp Ser Arg Leu Tyr Pro Gly AlaLeu Leu Val Val Asp Glu 65 70 75 80 Thr Leu Leu Glu Asn Asn Pro Thr LeuLeu Ala Val Asp Arg Ala Pro 85 90 95 Met Thr Tyr Ser Ile Asp Leu Pro GlyLeu Ala Ser Ser Asp Ser Phe 100 105 110 Leu Gln Val Glu Asp Pro Ser AsnSer Ser Val Arg Gly Ala Val Asn 115 120 125 Asp Leu Leu Ala Lys Trp HisGln Asp Tyr Gly Gln Val Asn Asn Val 130 135 140 Pro Ala Arg Met Gln TyrGlu Lys Ile Thr Ala His Ser Met Glu Gln 145 150 155 160 Leu Lys Val LysPhe Gly Ser Asp Phe Glu Lys Thr Gly Asn Ser Leu 165 170 175 Asp Ile AspPhe Asn Ser Val His Ser Gly Glu Lys Gln Ile Gln Ile 180 185 190 Val AsnPhe Lys Gln Ile Tyr Tyr Thr Val Ser Val Asp Ala Val Lys 195 200 205 AsnPro Gly Asp Val Phe Gln Asp Thr Val Thr Val Glu Asp Leu Lys 210 215 220Gln Arg Gly Ile Ser Ala Glu Arg Pro Leu Val Tyr Ile Ser Ser Val 225 230235 240 Ala Tyr Gly Arg Gln Val Tyr Leu Lys Leu Glu Thr Thr Ser Lys Ser245 250 255 Asp Glu Val Glu Ala Ala Phe Glu Ala Leu Ile Lys Gly Val LysVal 260 265 270 Ala Pro Gln Thr Glu Trp Lys Gln Ile Leu Asp Asn Thr GluVal Lys 275 280 285 Ala Val Ile Leu Gly Gly Asp Pro Ser Ser Gly Ala ArgVal Val Thr 290 295 300 Gly Lys Val Asp Met Val Glu Asp Leu Ile Gln GluGly Ser Arg Phe 305 310 315 320 Thr Ala Asp His Pro Gly Leu Pro Ile SerTyr Thr Thr Ser Phe Leu 325 330 335 Arg Asp Asn Val Val Ala Thr Phe GlnAsn Ser Thr Asp Tyr Val Glu 340 345 350 Thr Lys Val Thr Ala Tyr Arg AsnGly Asp Leu Leu Leu Asp Xaa Ser 355 360 365 Gly Ala Tyr Val Ala Gln TyrTyr Ile Thr Xaa Asp Glu Leu Ser Xaa 370 375 380 Xaa His Gln Gly Lys GluVal Leu Thr Pro Lys Ala Xaa Asp Arg Asn 385 390 395 400 Gly Gln Asp LeuThr Ala His Phe Thr Thr Ser Ile Pro Leu Lys Gly 405 410 415 Asn Val ArgAsn Leu Ser Val Lys Ile Arg Glu Xaa Thr Gly Leu Ala 420 425 430 Xaa XaaXaa Trp Arg Thr Val Tyr Glu Lys Thr Asp Leu Pro Leu Val 435 440 445 ArgLys Arg Thr Ile Ser Ile Trp Gly Thr Thr Leu Tyr Pro Gln Val 450 455 460Glu Asp Lys Val Glu Asn Asp 465 470 471 amino acids amino acid linearprotein 6 Met Ala Asn Lys Ala Val Asn Asp Phe Ile Leu Ala Met Asn TyrAsp 1 5 10 15 Lys Lys Lys Leu Leu Thr His Gln Gly Glu Ser Ile Glu AsnXaa Phe 20 25 30 Ile Lys Glu Gly Asn Gln Leu Pro Asp Glu Phe Val Val IleGlu Arg 35 40 45 Lys Lys Arg Ser Leu Ser Thr Asn Thr Ser Asp Ile Ser ValThr Ala 50 55 60 Thr Asn Asp Ser Arg Leu Tyr Pro Gly Ala Xaa Leu Val ValAsp Glu 65 70 75 80 Thr Leu Leu Glu Asn Asn Pro Thr Leu Leu Ala Val AspArg Ala Pro 85 90 95 Met Thr Tyr Ser Ile Asp Leu Pro Gly Leu Ala Ser SerAsp Ser Phe 100 105 110 Leu Gln Val Glu Asp Pro Ser Asn Ser Ser Val ArgGly Ala Xaa Asn 115 120 125 Asp Leu Leu Ala Lys Trp His Gln Asp Tyr GlyGln Val Asn Asn Val 130 135 140 Pro Ala Arg Met Gln Tyr Glu Lys Ile ThrAla Xaa Ser Met Glu Gln 145 150 155 160 Leu Lys Val Lys Phe Gly Ser AspPhe Glu Lys Thr Gly Asn Ser Leu 165 170 175 Asp Ile Asp Phe Asn Ser ValHis Ser Gly Glu Lys Gln Ile Gln Ile 180 185 190 Val Asn Phe Lys Gln IleTyr Tyr Thr Val Ser Val Asp Ala Val Lys 195 200 205 Asn Pro Gly Asp ValPhe Gln Asp Thr Val Thr Val Glu Asp Leu Lys 210 215 220 Gln Arg Gly IleSer Ala Glu Arg Pro Leu Val Tyr Ile Ser Ser Val 225 230 235 240 Ala TyrGly Arg Gln Val Tyr Leu Lys Leu Glu Thr Thr Ser Lys Ser 245 250 255 AspGlu Val Glu Ala Ala Phe Glu Ala Leu Ile Lys Gly Val Lys Val 260 265 270Ala Pro Gln Thr Glu Trp Lys Gln Ile Leu Asp Asn Thr Glu Val Lys 275 280285 Ala Val Ile Leu Gly Gly Asp Pro Ser Ser Gly Ala Arg Val Val Thr 290295 300 Gly Lys Val Asp Met Val Glu Asp Leu Ile Gln Glu Gly Ser Arg Phe305 310 315 320 Thr Ala Asp His Pro Gly Leu Pro Ile Ser Tyr Thr Thr SerPhe Leu 325 330 335 Arg Asp Asn Val Val Ala Thr Phe Gln Asn Ser Thr AspTyr Val Glu 340 345 350 Thr Lys Val Thr Ala Tyr Arg Asn Gly Asp Leu LeuLeu Asp Xaa Ser 355 360 365 Gly Ala Tyr Val Ala Gln Tyr Tyr Ile Thr XaaAsp Glu Leu Ser Xaa 370 375 380 Xaa Xaa Gln Gly Lys Xaa Val Leu Thr ProLys Ala Xaa Asp Arg Asn 385 390 395 400 Gly Gln Asp Leu Thr Ala His PheThr Thr Ser Ile Pro Leu Lys Gly 405 410 415 Asn Val Arg Asn Leu Ser ValLys Ile Arg Glu Xaa Thr Gly Leu Xaa 420 425 430 Xaa Xaa Xaa Xaa Arg ThrVal Tyr Glu Lys Thr Asp Leu Pro Leu Val 435 440 445 Arg Lys Arg Thr IleSer Ile Trp Gly Thr Thr Leu Tyr Xaa Gln Val 450 455 460 Glu Asp Lys ValGlu Asn Asp 465 470

What is claimed is:
 1. An altered and purified pneumolysin beingsubstantially non-toxic and being capable of eliciting a protectiveimmune response against pneumococcal bacteria in an animal, comprisingthe polypeptide of SEQ ID: 2 with one to three amino acid substitutionsin at least one of the regions 257-297, 367-397 and 427-437.
 2. Acomposition for eliciting an immune response against pneumococcalbacteria in an animal, comprising an adjuvant and the altered andpurified pneumolysin according to claim
 1. 3. An altered and purifiedpneumolysin being substantially non-toxic and being capable of elicitinga protective immune response against pneumococcal bacteria in an animal,comprising the polypeptide of SEQ ID: 2 with one to three amino acidsubstitutions in at least one of the regions 257-297, 367-397 and427-437, wherein said amino acid substitution is selected from the groupconsisting of: His₃₆₇→Arg; Cys₄₂₈→Gly; Cys₄₂₈→Ser; Trp₄₃₃→Phe;Glu₄₃₄→Gln; Trp₄₃₅→Phe; a combination of Asp₃₈₅→Asn and Cys₄₂₈→Gly; acombination of Trp₄₃₃→Phe and Asp₃₈₅→Asn; a combination of His₃₆₇→Argand Asp₃₈₅→Asn; and a combination of His₃₆₇→Arg, Asp₃₈₅→Asn andTrp₄₃₃→Phe.
 4. An altered and purified pneumolysin being substantiallynon-toxic and being capable of eliciting a protective immune responseagainst pneumococcal bacteria in an animal, comprising the polypeptideof SEQ ID NO: 2 with one to three amino acid substitutions, a said aminoacid substitution being of His₁₅₆ in SEQ ID NO:
 2. 5. A composition foreliciting an immune response against pneumococcal bacteria in an animal,comprising an adjuvant and the altered and purified pneumolysinaccording to claim
 4. 6. An altered and purified pneumolysin beingsubstantially non-toxic and being capable of eliciting a protectiveimmune response against pneumococcal bacteria in an animal, comprisingthe polypeptide of SEQ ID NO: 2 with one to three amino acidsubstitutions, a said amino acid substitution being of His₁₅₆ in SEQ IDNO: 2, wherein said at least one amino acid substitution is selectedfrom the group consisting of: His₁₅₆→Tyr; a combination of His₁₅₆→Tyrand Asp₃₈₅→Asn; and a combination of His₁₅₆→Tyr, Asp₃₈₅→Asn andTrp₄₃₃→Phe.
 7. An altered and purified pneumolysin being substantiallynon-toxic and being capable of eliciting a protective immune responseagainst pneumococcal bacteria in an animal, comprising the polypeptideof SEQ ID NO: 2 with one to three amino acid substitutions, a said aminoacid substitution being of His₁₅₆ in SEQ ID NO: 2, and furthercomprising an amino acid substitution in at least one of the regions257-297, 367-397 and 427-437 of SEQ ID NO:
 2. 8. An altered and purifiedpneumolysin having reduced haemolytic activity relative to wild-typepneumolysin and being capable of eliciting a protective immune responseagainst pneumococcal bacteria in an animal comprising a polypeptide ofSEQ ID NO: 2 with one to three amino acid substitutions in one of theregions 257-297, 367-397 and 427-437.
 9. An altered and purifiedpneumolysin having reduced haemoltyic activity relative to wild-typepneumolysin and reduced complement binding activity relative towild-type pneumolysin and being capable of eliciting a protective immuneresponse against pneumococcal bacteria in an animal, comprising apolypeptide of SEQ ID NO: 2 with one to three amino acid substitutionsin at least one of the regions 257-297, 367-397 and 427-437.
 10. Analtered and purified pneumolysin having reduced haemolytic activityrelative to wild-type pneumolysin and being capable of eliciting aprotective immune response against pneumococcal bacteria in an animalcomprising a polypeptide of SEQ ID NO: 2 with one to three amino acidsubstitutions in one of three regions 257-297, 367-397 and 427-437,wherein one of said amino acid substitutions is Trp₄₃₅→Phe.
 11. Analtered and purified pneumolysin having reduced haemolytic activityrelative to wild-type pneumolysin and being capable of eliciting aprotective immune response against pneumococcal bacteria in an animalcomprising a polypeptide of SEQ ID NO: 2 with one to three amino acidsubstitutions in one of three regions 257-297, 367-397 and 427-437,wherein said amino acid substitutions comprise Trp₄₃₃→Phe andAsp₃₈₅→Asn.
 12. An altered and purified pneumolysin being substantiallynon-toxic and being capable of eliciting a protective immune responseagainst pneumococcal bacteria in an animal, comprising the polypeptideof SEQ ID NO: 2 with one to three amino acid substitutions in at leastone of the regions 257-297, 367-397 and 427-437, wherein one of saidamino acid substitutions is His_(367→)Arg.
 13. An altered and purifiedpneumolysin being substantially non-toxic and being capable of elicitinga protective immune response against pneumococcal bacteria in an animal,comprising the polypeptide of SEQ ID NO: 2 with one to three acidsubstitutions in at least one of the regions 257-297, 367-397 and427-437, wherein one of said amino acid substitutions is Cys_(428→)Gly.14. An altered and purified pneumolysin being substantially non-toxicand being capable of eliciting a protective immune response againstpneumococcal bacteria in an animal, comprising the polypeptide of SEQ IDNO: 2 with one to three amino acid substitutions in at least one of theregions 257-297, 367-397 and 427-437, wherein one of said amino acidsubstitutions is Cys_(428→)Ser.
 15. An altered and purified pneumolysinbeing substantially non-toxic and being capable of eliciting aprotective immune response against pneumococcal bacteria in an animal,comprising the polypeptide of SEQ ID NO: 2 with one to three amino acidsubstitutions in at least one of the regions 257-297, 367-397 and427-437, wherein one of said amino acid substitutions is Glu₄₃₄→Gln. 16.An altered and purified pneumolysin being substantially non-toxic andbeing capable of eliciting a protective immune response againstpneumococcal bacteria in an animal, comprising the polypeptide of SEQ IDNO:2 with one to three amino acid substitutions in at least one of theregions 257-297, 367-397 and 427-437, wherein one of said amino acidsubstitutions is Trp₄₃₅→Phe.
 17. An altered and purified pneumolysinbeing substantially non-toxic and being capable of eliciting aprotective immune response against pneumococcal bacteria in an animal,comprising the polypeptide of SEQ ID NO: 2 with one to three amino acidsubstitutions in at least one of the regions 257-297, 367-397 and427-437, wherein said amino acid substitutions comprise Asp₃₈₅→Asn andCys_(428→)Gly.
 18. An altered and purified pneumolysin beingsubstantially non-toxic and being capable of eliciting a protectiveimmune response against pneumococcal bacteria in an animal, comprisingthe polypeptide of SEQ ID NO:2 with one to three amino acidsubstitutions in at least one of the regions 257-297, 367-397 and427-437, wherein one of said amino acid substitutions comprisesTrp₄₃₃→Phe and Asp₃₈₅→Asn.
 19. An altered and purified pneumolysin beingsubstantially non-toxic and being capable of eliciting a protectiveimmune response against pneumococcal bacteria in an animal, comprisingthe polypeptide of SEQ ID NO:2 with one to three amino acidsubstitutions in at least one of the regions 257-297, 367-397 and427-437, wherein said one to three amino acid substitutions compriseHis_(367→)Arg and Asp₃₈₅→Asn.
 20. An altered and purified pneumolysinbeing substantially non-toxic and being capable of eliciting aprotective immune response against pneumococcal bacteria in an animal,comprising the polypeptide of SEQ ID NO: 2 with one to three amino acidsubstitutions, a said amino acid substitution being His₁₅₆ in SEQ IDNO:2, and wherein said one to three amino acid substitutions compriseHis₁₅₆→Tyr and Asp₃₈₅→Asn.
 21. An altered and purified pneumolysin beingsubstantially non-toxic and being capable of eliciting a protectiveimmune response against pneumococcal bacteria in an animal, comprisingthe polypeptide of SEQ ID NO:2 with one to three amino acidsubstitutions, a said amino acid substitution being His₁₅₆ in SEQ IDNO:2, and wherein said one to three amino acid substitutions compriseHis₁₅₆→Tyr; Asp₃₈₅→Asn and Trp₄₃₃→Phe.